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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 has a Stokes radius of 7.2 in buffers of high ioninc strength, suggesting a molecular weight in the range 145,000 to 195,000. The polypeptide bands observed on gel electrophoresis in dodecyl sulfate have apparent molecular weights of 78,000 and 69,000 (and possibly another 27,000) in equimolar amounts. In buffers of low ionic strength, the enzyme appears to form large aggregates and even precipitates, with about 90% loss of activity. A nuclease activity co-purifies with the PBS2 DNA polymerase and shows similar responses to changes in pH, MgCl2, N-ethylmaleimide, temperature, and dextran sulfate levels. The nuclease produces deoxyribonucleoside 5'monophosphates from denatured DNA containing thymine or uracil. No endonuclease activity is detectable on supercoiled DNA. The inhibition of nuclease activity by added deoxyribonucleoside triphosphates, the DNA-dependent turnover of triphosphates, to free monophosphates during DNA polymerization, the inhibition of nuclease activity by 3'-phosphates on the DNA template-primer, and the pattern of digestion of 5'-[32P]phosphate-labeled DNA all indicate that the PBS2 DNA polymerase-associated hydrolytic activity is a 3' leads to 5'-exonuclease.
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PMID:Characterization of the Bacillus subtilis bacteriophage PBS2-induced DNA polymerase and its associated exonuclease activity. 10 39

The DNA polymerase of early embryos of Drosophila melanogaster has been purified to near-homogeneity. The purified enzyme gave a single, catalytically active protein band after polyacrylamide gel electrophoresis, under nondenaturing conditions. Four polypeptides with molecular weights 43,000, 46,000, 58,000, and 148,000 were resolved when this band was electrophoresed under denaturing conditions. At high ionic strengths, the DNA polymerase had a sedimentation coefficient of 8.7 S, a Stokes radius of 78 A and frictional ratio of 1.81, parameters that yield a molecular weight of 280,000. The purified DNA polymerase possessed no detectable endo- or exodeoxyribonuclease, ATPase, or RNA polymerase activity. Using an "activated" DNA template-primer, the enzyme had a pH optimum of 8.5. It was stimulated by (NH4)2SO4, KCl, and to a lesser extent, NaCl. A divalent metal cation was absolutely required; MgCl2 stimulating activity 7-fold more than MnCl2. It was inhibited by low concentrations of N-ethylmaleimide and Aphidicolon. Thus the DNA polymerase of D. melanogaster resembles most closely the alpha-DNA polymerases that have been purified from mammalian cells.
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PMID:A high molecular weight DNA polymerase from Drosophila melanogaster embryos. Purification, structure, and partial characterization. 11 15

Simian virus 40 (SV40) nucleoprotein complexes were extracted from nuclei of infected monkey cells and fractionated on neutral sucrose density gradients. Complexes which contained replicating SV40 DNA (95S) separated well from those containing closed circular supercoiled viral DNA (75S). DNA polymerase activity was associated with the replicating nucleoprotein complexes but not with the slower sedimenting complexes. This DNA polymerase activity coprecipitated with the nucleoprotein complexes in the presence of MgCl2 and remained associated with the 95S complexes. This DNA polymerase activity has been identified as primarily DNA polymerase alpha on the basis of its sedimentation behavior, optimum salt concentration, and sensitivity to N-ethylmaleimide. DNA polymerase gamma activity was also detected in the complexes, but DNA polymerase beta was not associated with the complexes.
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PMID:DNA polymerase alpha is associated with replicating SV40 nucleoprotein complexes. 20 60

Distinct DNA polymerase activities have been found in the cytoplasmic and nuclear fractions of a baby hamster kidney cell line. They were separated by chromatography on DEAE-cellulose and partially purified by ammonium sulfate fractionation, DNA - cellulose and linear sucrose gradients. The cytoplasmic DNA polymerase exhibited an S-coefficient of 6.95 S in 0.15 M NaCl and its activity was highly sensitive to inhibition by N-ethylmaleimide and elevated temperatures, regardless of the presence of DNA template or other cofactors. It was stimulated by monovalent salts in the order of NH4 Cl greater than KCl greater than NaCl greater than CsCl greater than LiCl (inhibitory). The DNA polymerase extracted from nuclei sedimented with an S-value of 3.47 S, was resistant to inactivation by N-ethylmaleimide, and maximally stimulated by NaCl, while also being inhibited by LiCl. For optimal activity, both DNA polymerase activities required a divalent cation, with MgCl2 being more effective than MnCl2. Although the optimal pH values for the two enzyme activities differed slightly, glycine - NaOH buffer induced an alkaline shift of 1.5 pH units in the optimum of both enzymes. This was accompanied by an increase in the effectiveness of MnCl2 relative to MgCl2 for the cytoplasmic DNA polymerase.
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PMID:Differentiation and characterization of the cytoplasmic and nuclear deoxyribonucleic acid polymerases from baby hamster kidney cells. 24 Apr 21

alpha and beta DNA polymerases (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) were isolated from nuclear and cytoplasmic fractions of rat livers exposed to a carcinogenic regimen with the hepatocarcinogen N-2-fluorenylacetamide and from 24-hr regenerating liver. The fidelity of polymerization of these enzymes was compared by determining the incorporation of noncomplementary deoxyribonucleoside triphosphates (misincorporation) on a poly(dA-dT).poly(dA-dT) template, with MnCl2 and MgCl2 as divalent cations. Our initial studies indicate that the cytoplasmic alpha polymerases from carcinogen-exposed rat livers were strikingly error-prone whereas the nuclear and cytoplasmic beta polymerases retained their fidelity throughout the feeding cycles. The misincorporation was significantly accentuated by MnCl2 compared with that obtained with MgCl2 as divalent cation. The products were sensitive to pancreatic DNase I digestion, indicating that the noncomplementary bases had been incorporated by the polymerization process. Nuclear alpha polymerase showed some degree of infidelity but less than that of cytoplasmic alpha polymerase.
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PMID:Decreased fidelity of DNA polymerase activity during N-2-fluorenylacetamide hepatocarcinogenesis. 28 2

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A--T)n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA)n . (dT)10 and (rA)n . (dT)10 . (20:1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA)n . (dT)10 and somewhat less for (dA)n . (dT)10. The apparent Km for the primer (rA)n . (dT)10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent Km for dTTP. The influence of polyamines on both the 5' leads to 3' and 3' leads to 5' nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A--T)n and T7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of the activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.
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PMID:Influence of polyamines on the activity of DNA polymerase I from Escherichia coli. 36 Oct 88

Sphingomyelin, phosphatidylethanolamine and lecithin at concentrations which destabilize the DNA helix enhance the DNA polymerase activity, but sphingosine and phosphatidylserine have only a moderate effect. MgCl2 was shown to modify the action of sphingomyelin on the DNA polymerase activity. The role of phospholipids in the DNA replicating process was analyzed.
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PMID:Effect of phospholipids on the activity of DNA polymerase I from E. coli. 76 89

The DNA replication system of S-phase HeLa nuclei has been dissociated by cautious extraction at 0 degrees C with 0.25 M NaCl. Replicase activity has been reestablished by recombination of the fractions and reduction of the salt concentration. The reconstituted system, like the starting nuclei, depended on ATP, 4dNTP, MgCl2, the proper ionic strength and the soluble cytoplasmic protein fraction. The activity of the nuclear extract showed a cell cycle dependency and was elevated in the nuclei of cells at the G1 leads to S boundary. In the presence of Mg2+ the major activity of the nuclear extract precipitated during dialysis to reduce the salt concentration; this precipitate exhibited DNA polymerase alpha activity. Chromatography of the active extracts over phosphocellulose separated the replicase supporting factors into three fractions. The major activity eluted in the fraction containing the DNA polymerase alpha activity; the other two active fractions were devoid of polymerase activity. The fraction containing DNA polymerase alpha from the nuclear extracts supported DNA replicase activity in salt-extracted nuclei whereas an equivalent level of DNA polymerase alpha from the cytoplasm was not effective. The data suggest that the DNA polymerase alpha of the salt extracts of S-phase nuclei is either different than the cytoplasmic enzyme or is associated with some essential replicase-supporting factor.
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PMID:Dissociation and reconstitution of the DNA replicase system of HeLa cell nuclei. 94 95

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.
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PMID:Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos. 133 52

We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng.microliters-1 and high MgCl2 levels was especially important for reproducibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.
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PMID:DNA amplification fingerprinting of bacteria. 136 11


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